Protein prenylation is a post-translational lipid modification that involves the covalent attachment of isoprenoid groups onto cysteine residues at or near the carboxyl termini of proteins. The attachment of a lipophilic isoprenoid group to proteins is believed to increase their hydrophobicity, allowing otherwise hydrophilic proteins to associate with membranes. Up to 0.5% of total cellular proteins are estimated to be prenylated. Known prenylated proteins include small GTP-binding proteins of the Ras superfamily, nuclear lamins, the yeast mating pheromone .alpha.-factor and trimeric G proteins. These proteins are engaged in a variety of cellular processes, which include the control of cell growth, signal transduction, cytokinesis, and intracellular membrane traffic.
Two different isoprenoid groups, farnesyl (15 carbons) and geranylgeranyl (20 carbons), are post-translationally attached to proteins. Farnesyl is added to proteins that terminate in a CAAX motif (where C is cysteine, A is an aliphatic amino acid, and X can be methionine, cysteine, alanine, glutamine, phenylalanine, or serine), while geranylgeranyl is transferred onto proteins that end in CAAL (where L is leucine), CC, or CXC motifs (X is any amino acid). Most known prenylated proteins are geranylgeranylated.
Farnesyl and geranylgeranyl groups are attached to proteins from all-trans farnesyl diphosphate (FPP) and all-trans geranylgeranyl diphosphate (GGPP), respectively. These lipid precursors are intermediates in the isoprenoid biosynthetic pathway. This pathway consists of a series of reactions by which mevalonate is converted into a diverse family of lipophilic molecules that contain a repetitive five-carbon structure. The isoprenoids are subsequently incorporated into a large number of end products, which includes: sterols, ubiquinones, dolichols, tRNAs, and prenylated proteins.
FPP is the product of the farnesyl diphosphate synthase reaction. This enzyme, which is the most abundant and widely occurring prenyltransferase, catalyzes the formation of FPP by the sequential addition of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP), and geranyl diphosphate (GPP). In some organisms, GGPP is synthesized by a GGPP synthase (GGPPS) that catalyzes stepwise additions of IPP to DMAPP, GPP, and FPP. This type of GGPP synthase activity has been detected in mammalian tissue. However, eukaryotic geranylgeranyl diphosphate synthases are known that synthesize GGPP by the addition of a single molecule of IPP to FPP. But, due to its low activity and the problems in separating this enzyme from FPP synthase, its purification has proven to be difficult.
GGPP is the substrate for two different protein prenyltransferases, the type I (GGTase-I) and type II (GGTase-II) geranylgeranyl transferases. GGTase-I catalyzes the transfer of a geranylgeranyl group from GGPP onto proteins that terminate in a CAAL motif, while GGTase-II attaches geranylgeranyl to terminal CC or CXC residues. Its protein substrates include members of the Ras family of small GTP-binding proteins.
GGPP and FPP are important intermediates in the formation of a variety of derivatives which have important uses in the production of anti-cancer compounds, anti-tumor compounds, anti-cholesterol compounds and anti-ulcer compounds. For example, GGPP and FPP can be used in the prenylation of ras oncogene protein to inhibit neoplastic transformation. Taxol, a potent anti-cancer agent, is a GGPP derivative for which there is currently a lack of cost-effective, biosynthetic methods of production. Therefore, isolation of GGPP synthases for use in modulating GGPP and FPP biosynthetic pathways is both desirable and commercially valuable.
Although prior investigators have identified GGPP synthases in organisms such as bacteria, archaebacteria, rodents, bovines and filamentous fungi, GGPP synthases have been difficult to isolate. Prior to the present invention, a GGPP synthase has never been identified in yeast. Therefore, there is a need for the isolation of genes which encode eukaryotic GGPP synthases for use in the production of GGPP in large quantities, in a cost-effective manner.